Method for measuring in vivo mutation frequency at an endogenous gene locus

a technology of endogenous gene and which is applied in the field of in vivo mutation frequency measurement at an endogenous gene locus, can solve the problems of expensive breeding programs, absence or reduction of membrane expression of gpi-linked proteins in peripheral blood cells, etc., and achieves the effect of fast, reliable and accura

Active Publication Date: 2007-11-29
LITRON LAB
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  • Abstract
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AI Technical Summary

Benefits of technology

[0017]The present invention identifies procedures that can be employed for an automated in vivo mutation assay that can be used to evaluate agents (e.g., chemical or physical agents) for genetic toxicity. It can also be used to assess the magnitude and / or consequences of occupational, accidental, other unintentional exposure scenarios. The procedure is fast, reliable, and accurate, and can be performed without the need for transgenic animal models or cell culture work.

Problems solved by technology

While some are based on colony formation and therefore require time-consuming tissue culture work after target cells have been harvested, others require expensive breeding programs to supply rodents with a specific genotype.
Mutations giving rise to non-functional pig-a product result in the absence or the reduced membrane expression of GPI-linked proteins in peripheral blood cells.

Method used

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  • Method for measuring in vivo mutation frequency at an endogenous gene locus
  • Method for measuring in vivo mutation frequency at an endogenous gene locus
  • Method for measuring in vivo mutation frequency at an endogenous gene locus

Examples

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example 1

Differential Labeling of Erythrocytes, Reticulocytes, Nucleated Cells, Platelets, and Mutant Erythrocytes

[0062]In this example, a three-color procedure for differentially labeling and scoring pig-a cells is demonstrated.

[0063]Whole blood specimens (EDTA as anticoagulant) were obtained from the tail vein of vehicle- or ethyl-N-nitrosourea (ENU) treated rats (100 mg / kg / day for three days at 48 hr intervals; blood collected several weeks after the final administration). These specimens were centrifuged at low speed at least two times to significantly reduce the number of platelets. Thereafter, cells were contacted with anti-CD59-PE to differentially label mutant and non-mutant erythrocytes, and biotinylated anti-CD61 followed by streptavidin-Cy5-PE as a platelet-specific label used to exclude these particles from analysis. After washing steps to remove unbound antibodies and unbound steptavidin-Cy5-PE, cells were resupsended in a thiazole orange solution (0.1 μg / ml). Specimens were inc...

example 2

Thresholding Technique Provides Rapid Assessment of Reticulocytes for pig-a Mutant Phenotype

[0066]The thresholding technique is based on the fact that the vast majority of unfractionated blood cells are mature erythrocytes.

[0067]With appropriate staining (e.g., thiazole orange, SYTO 13 dye, SYTO 83 dye, RNASelect, or anti-CD71), it is possible to distinguish reticulocytes from their more mature counterparts.

[0068]In this case, it is possible to set the threshold parameter to the nucleic acid dye-associated fluorescence channel (e.g., FL1 for thiazole orange), as opposed to the more common forward scatter trigger. When set sufficiently high (that is, above the fluorescence intensity of mature erythrocytes), this can restrict analysis to reticulocytes.

[0069]Even so, fluorescence thresholding alone does not improve reticulocyte interrogation rates. Rather, it must be coupled with the preparation of very high density specimens. When these two adjustments are made, much quicker data acqu...

example 3

Selective Lysis of Wild-Type Erythrocytes Enhances the Speed of Mutant Phenotype Scoring

[0071]A second approach for enhancing the rate at which erythrocytes can be evaluated for the pig-a mutant phenotype is based on selective cell lysis.

[0072]To demonstrate this technique, female Sprague-Dawley rats were either untreated, or exposed to ENU at 100 mg / kg / day or 7,12-dimethyl-1,2-benz[a] anthracene (DMBA) at 40 mg / kg / day. Treatment occurred on three days (M, W, F), and tail vein blood was collected 6 weeks thereafter into EDTA-containing solutions. Blood cells were washed two times and are then stained with three fluorescent reagents according to the present invention. Specifically, specimens were contacted with anti-CD59-FITC to differentially label mutant and non-mutant erythrocytes, biotinylated anti-CD61 followed by streptavidin Cy5-PE as a platelet-specific label used to exclude these particles from analysis, and finally SYTO 83 dye to differentially stain mature erythrocytes, re...

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Abstract

The present invention relates a method for the enumeration of in vivo gene mutation. The method utilizes differential staining of GPI-anchor deficient erythrocyte populations to distinguish between wild-type and pig-a gene mutants. Quantitative analyses can be conducted on erythrocytes and / or reticulocytes, and is based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of mutant erythrocytes or reticulcoytes relative to the number of total erythrocytes or reticulocytes can be used to assess the DNA-damaging potential of an exogenous chemical agent, the DNA-damaging potential of an exogenous physical agent, the effects of an exogenous agent which can modify endogenously-induced DNA damage, and the effects of an exogenous agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.

Description

[0001]This application claims the priority benefit of provisional U.S. Patent Application Ser. No. 60 / 808,445, filed May 25, 2006, which is hereby incorporated by reference in its entirety.[0002]This work was supported in part by a grant from the National Cancer Institute under grant number R43 CA106063. The U.S. government may retain certain rights in this invention.FIELD OF THE INVENTION[0003]This invention relates to an analytical method for measuring the in vivo mutation frequency caused by an exogenous agent to which a mammal has been exposed, either intentionally or unintentionally.BACKGROUND OF THE INVENTION[0004]DNA damage can result in mutation, and this is a primary mechanism by which cancers arise. These events have also been implicated in diseases such as atherosclerosis, and processes such as aging. Therefore, there is an important need for sensitive methods which are capable of identifying chemical or physical agents that can mutate DNA. Given the tremendous cost of lo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K49/00C12Q1/00G01N33/567
CPCG01N33/80G01N2333/70557G01N2333/70582G01N2333/70596Y10S435/973Y10T436/25125Y10T436/107497Y10T436/101666Y10S436/811Y10T436/143333
Inventor DERTINGER, STEPHEN D.
Owner LITRON LAB
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